DOCSLIB.ORG

(EMSA) for Measuring Dissociation Constants of Protein‐RNA Complexes

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
(EMSA) for Measuring Dissociation Constants of Protein‐RNA Complexes Label-Free Electrophoretic Mobility Shift Assay (EMSA) for Measuring Dissociation Constants of Protein-RNA Complexes Minguk Seo,1 Li Lei,1 and Martin Egli1,2 1Department of Biochemistry, School of Medicine, Vanderbilt University, Nashville, Tennessee 2Corresponding author: [email protected] The electrophoretic mobility shift assay (EMSA) is a well-established method to detect formation of complexes between proteins and nucleic acids and to determine, among other parameters, equilibrium constants for the interaction. Mixtures of protein and nucleic acid solutions of various ratios are analyzed via polyacrylamide gel electrophoresis (PAGE) under native conditions. In general, protein–nucleic acid complexes will migrate more slowly than the free nucleic acid. From the distributions of the nucleic acid components in the observed bands in individual gel lanes, quantitative parameters such as the dissociation constant (Kd) of the interaction can be measured. This article describes a simple and rapid EMSA that relies either on precast commercial or handcast polyacrylamide gels and uses unlabeled protein and nucleic acid. Nucleic acids are instead detected with SYBR Gold stain and band intensities established with a standard gel imaging system. We used this protocol specifically to determine Kd values for complexes between the PAZ domain of Argonaute 2 (Ago2) enzyme and native and chemically modified RNA oligonucleotides. EMSA- based equilibrium constants are compared to those determined with isothermal titration calorimetry (ITC). Advantages and limitations of this simple EMSA are discussed by comparing it to other techniques used for determination of equilibrium constants of protein-RNA interactions, and a troubleshooting guide is provided. C 2018 by John Wiley & Sons, Inc. Keywords: Ago2 r electrophoresis r equilibrium constant r protein–nucleic acid interaction r RNA How to cite this article: Seo, M., Lei, L., & Egli, M. (2019). Label-free electrophoretic mobility shift assay (EMSA) for measuring dissociation constants of protein-RNA complexes. Current Protocols in Nucleic Acid Chemistry, 76, e70. doi: 10.1002/cpnc.70 Nucleic acid–protein interactions are ubiquitous and absolutely essential in biological BASIC PROTOCOL information transfer, including replication, transcription, repair, and RNA metabolism (Cusack et al., 2017). Gel electrophoresis using polyacrylamide gels (PAGE; Andrus & Kuimelis, 2001a) and the electrophoretic mobility shift assay (EMSA) remain im- portant and are widely used approaches for detecting nucleic acid–protein interac- tions and determining the stability of individual complexes (Alves & Cunha, 2012; Chen, 2011; Fried, 1989; Hellman & Fried, 2007). This protocol details the steps for establishing the equilibrium dissociation constant Kd of an RNA-protein com- plex by EMSA. Solutions of RNA and protein are mixed in different ratios, and the Seo et al. Current Protocols in Nucleic Acid Chemistry e70, Volume 76 1of12 Published in Wiley Online Library (wileyonlinelibrary.com). doi: 10.1002/cpnc.70 C 2018 John Wiley & Sons, Inc. binding reactions are then separated by non-denaturing PAGE (https://tools.thermofisher. com/content/sfs/brochures/1601945-Protein-Interactions-Handbook.pdf). In general, a protein-RNA complex will migrate more slowly through the gel matrix relative to the RNA alone, thus causing a shift on the gel (e.g., Yang et al., 1999). Individual bands can be visualized by end-labeling the RNA radioactively (32P) or by using fluorescent or chemiluminescent probes in combination with a gel imager. However, in our assay, we have used non-labeled RNA together with the SYBR Gold stain (Tuma et al., 1999) for detecting and quantifying bands. From the distribution of the RNA component among the protein-RNA and RNA bands in individual lanes on the gel, one can determine the equilibrium dissociation constant Kd of the complex in a straightforward manner. An approximate value for the equilibrium constant can be obtained by finding the concen- tration of protein at which roughly half the nucleic acid component is bound and half remains free. A more precise way to determine Kd is to plot individual fractions of the nucleic acid bound in each reaction versus the concentration of protein and then perform a nonlinear regression (Heffler, Walters, & Kugel, 2012) using free or commercially available software. Human Argonaute 2 (Ago2) is an 859–amino acid protein that lies at the heart of the RNA- induced silencing complex (RISC; Sheu-Gruttadauria & MacRae, 2017). The protein binds small interfering RNA (siRNA) duplexes consisting of 21mer guide (antisense) and passenger (sense) strands with 3-terminal dinucleotide overhangs. Ago2 contains multiple domains, including the MID (binds 5-end of guide siRNA), PIWI (harbors the endonuclease active site), PAZ (binds 3-end of guide siRNA), and two linkage domains. The passenger strand is eventually cleaved or discarded and the target RNA loaded into Ago2 opposite the guide strand, resulting in cleavage of the former by the PIWI domain via a dual-metal ion mechanism (Watts & Corey, 2012; Wilson & Doudna, 2013). RISC contains other Ago proteins besides Ago2 (i.e., Ago1, Ago3, and Ago4), and while all are associated with micro RNAs (miRNAs), only Ago2 exhibits endonuclease activity (Meister et al., 2004). Chemically modified siRNAs are widely explored as therapeutics for treatment of a range of diseases (Crooke, Witztum, Bennett, & Baker, 2018; Shen & Corey 2018). ONPATTRO (Patisiran) is a modified siRNA formulated in lipids and manufactured by Alnylam Pharmaceuticals Inc. (Cambridge, MA) that was approved by the US FDA in August of 2018 for the treatment of polyneuropathy of hereditary transthyretin-mediated amyloidosis in adults (Sheridan, 2017). The PAZ domain of Ago2 binds the last two nucleotides of guide siRNA and assists in separating guide and passenger siRNA (Elkayam et al., 2012; Ma, Ye, & Patel, 2004; Schirle & MacRae, 2012; Fig. 1). The identities of the 3-overhanging nucleotides in the siRNA guide strand and their chemical modification affect the interaction with Ago2 PAZ and silencing activity (Alagia et al., 2018; Kandeel et al., 2014). We relied on the EMSA protocol presented here to determine equilibrium dissociation constants Kd of the interactions between native and chemically modified RNAs and the human Ago2 PAZ domain. The following procedures outline the mixing of RNA and protein solutions of defined concentrations in various ratios to establish the binding reactions, the preparation of polyacrylamide gels to run EMSAs for analyzing protein-RNA binding, staining and imaging of gels to measure the intensities of individual bands and the distribution of RNA between the free and protein-bound forms, and the determination of Kd values us- ing standard software to compute nonlinear regressions. However, neither the synthesis of RNA oligonucleotides nor the expression and purification of the Ago2 PAZ domain are described in detail. Briefly, RNAs were provided by Alnylam Pharmaceuticals Inc. (Cambridge, MA; native oligoribonucleotides) or AM Biotechnologies LLC (Houston, TX; 2-O-Me/3-phosphorodithioate [PS2; both non-bridging phosphate oxygen atoms Seo et al. replaced with sulfur] oligoribonucleotide, hereafter referred to as MS2-modified RNA). 2of12 Current Protocols in Nucleic Acid Chemistry Figure 1 Overlay of crystal structures of human Ago1- and Ago2-PAZ in complex with RNA oligonucleotides (A) and alignment of the human Ago1 (top line) and Ago2 (bottom line) sequences (B). Only a portion of the 859 amino acids is included; the consensus sequence is shown in the middle line with PAZ domain residues highlighted: 225 to 369 (Ago1) and 227 to 371 (Ago2). The PAZ domain binds the 3-end of guide siRNA. The models shown are from crystal structures of full-length Ago2 bound either to duplex RNA (Schirle & MacRae, 2012) or an miRNA single strand (Elkayam et al., 2012 ). In both cases, only PAZ domain residues 226 to 351 were included in the illustration. In both crystal structures, only portions of the 3-half of the RNA guide strand were visible in the electron density. The third structure depicted is of a complex between a separately expressed human Ago1 PAZ domain (residues 224 to 349 were resolved in the electron density) and an RNA nonamer (Ma et al., 2004). Coordinates were retrieved from the Protein Data Bank (PDB; https://www.rcsb.org/; Berman et al., 2000), and the structural figure in (A) was generated with the program UCSF Chimera (Pettersen et al., 2004). Native RNAs were synthesized by standard or adapted solid phase phosphoramidite syn- thesis, purified (Andrus & Kuimelis, 2001b) by high-performance liquid chromatography (HPLC; Sinha & Jung, 2015), and desalted (Andrus & Kuimelis, 2001c). Identities and purities of all oligonucleotides were confirmed by electrospray ionization mass spectrom- etry (ESI-MS) and ion-exchange HPLC (IEX-HPLC), respectively. The MS2-modified RNA was synthesized and purified as reported in Yang (2017). An expression system for human Ago2 PAZ (amino acids 227 to 371; Fig. 1B) was generated by gene synthesis (GenScript Inc.) and subcloning into the pHD116 plasmid. The plasmid was transformed into E. coli BL21 (DE3) Gold cells using standard procedures. After expression the Seo et al. 3of12 Current Protocols in Nucleic Acid Chemistry protein was first purified by nickel affinity chromatography and, following cleavage of the His6 tag by PreScission protease (GE Healthcare), by ion exchange and gel filtra- tion chromatography. The
Load more

Recommended publications
  • Not Dicer but Argonaute Is Required for a Microrna Production
    Cell Research (2010) 20:735-737. npg © 2010 IBCB, SIBS, CAS All rights reserved 1001-0602/10 $ 32.00 RESEARCH HIGHLIGHT www.nature.com/cr A new twist in the microRNA pathway: Not Dicer but Argonaute is required for a microRNA production Gabriel D Bossé1, Martin J Simard1 1Laval University Cancer Research Centre, Hôtel-Dieu de Québec (CHUQ), Quebec City, Québec G1R 2J6, Canada Cell Research (2010) 20:735-737. doi:10.1038/cr.2010.83; published online 15 June 2010 Found in all metazoans, microRNAs A Canonical pathway B Ago2-dependent pathway or miRNAs are small non-coding RNA Nucleus Cytoplasm Nucleus Cytoplasm of ~22 nucleotides in length that com- Exp.5 Exp.5 pletely reshaped our understanding of gene regulation. This new class of gene pre-miR-451 regulator is mostly transcribed by the pre-miRNA RNA polymerase II producing a long stem-loop structure, called primary- or Ago2 pri-miRNA, that will first be processed Ago2 Dicer in the cell nucleus by a multiprotein TRBP complex called microprocessor to gen- erate a shorter RNA structure called Ago2 RISC precursor- or pre-miRNA. The precisely Ago2 RISC processed pre-miRNA will next be ex- ported into the cytoplasm by Exportin 5 and loaded onto another processing machine containing the ribonuclease III enzyme Dicer, an Argonaute protein Ago2 Ago2 and other accessory cellular factors mRNA mRNA (Figure 1A; [1]). Dicer will mediate the Translation inhibition Translation inhibition cleavage of the pre-miRNA to form the mature miRNA that will then be bound Figure 1 (A) Canonical microRNA biogenesis. In mammals, the pre-miRNA is by the Argonaute protein to form, most loaded onto a multiprotein complex consisting minimally of Dicer, Tar RNA Bind- likely with other cellular factors, the ef- ing Protein (TRBP) and Ago2.
    [Show full text]
  • EIF2C2 Monoclonal Antibody (M01), Clone 2E12-1C9
    EIF2C2 monoclonal antibody (M01), clone 2E12-1C9 Catalog # : H00027161-M01 規格 : [ 100 ug ] List All Specification Application Image Product Mouse monoclonal antibody raised against a full length recombinant Western Blot (Cell lysate) Description: EIF2C2. Immunogen: EIF2C2 (AAH07633.1, 483 a.a. ~ 859 a.a) full-length recombinant protein with GST tag. MW of the GST tag alone is 26 KDa. Sequence: MPIQGQPCFCKYAQGADSVEPMFRHLKNTYAGLQLVVVILPGKTPVYAE VKRVGDTVLGMATQCVQMKNVQRTTPQTLSNLCLKINVKLGGVNNILLP enlarge QGRPPVFQQPVIFLGADVTHPPAGDGKKPSIAAVVGSMDAHPNRYCATV Western Blot (Transfected RVQQHRQEIIQDLAAMVRELLIQFYKSTRFKPTRIIFYRDGVSEGQFQQV lysate) LHHELLAIREACIKLEKDYQPGITFIVVQKRHHTRLFCTDKNERVGKSGNIP AGTTVDTKITHPTEFDFYLCSHAGIQGTSRPSHYHVLWDDNRFSSDELQI LTYQLCHTYVRCTRSVSIPAPAYYAHLVAFRARYHLVDKEHDSAEGSHTS GQSNGRDHQALAKAVQVHQDTLRTMYFA Host: Mouse enlarge Reactivity: Human Western Blot (Recombinant Isotype: IgG1 Kappa protein) Quality Control Antibody Reactive Against Recombinant Protein. Immunofluorescence Testing: enlarge Immunohistochemistry (Formalin/PFA-fixed paraffin- embedded sections) Western Blot detection against Immunogen (68.03 KDa) . Storage Buffer: In 1x PBS, pH 7.4 enlarge Storage Store at -20°C or lower. Aliquot to avoid repeated freezing and thawing. Instruction: Sandwich ELISA (Recombinant protein) MSDS: Download Interspecies Mouse (100); Rat (99) Antigen Sequence: enlarge Datasheet: Download ELISA Publication Reference RNAi Knockdown (Antibody validated) 1. TDP-43 aggregation induced by oxidative stress causes global mitochondrial imbalance
    [Show full text]
  • Mir-125 in Normal and Malignant Hematopoiesis
    Leukemia (2012) 26, 2011–2018 & 2012 Macmillan Publishers Limited All rights reserved 0887-6924/12 www.nature.com/leu SPOTLIGHT REVIEW MiR-125 in normal and malignant hematopoiesis L Shaham1,2, V Binder3,4,NGefen1,5, A Borkhardt3 and S Izraeli1,5 MiR-125 is a highly conserved microRNA throughout many different species from nematode to humans. In humans, there are three homologs (hsa-miR-125b-1, hsa-miR-125b-2 and hsa-miR-125a). Here we review a recent research on the role of miR-125 in normal and malignant hematopoietic cells. Its high expression in hematopoietic stem cells (HSCs) enhances self-renewal and survival. Its expression in specific subtypes of myeloid and lymphoid leukemias provides resistance to apoptosis and blocks further differentiation. A direct oncogenic role in the hematopoietic system has recently been demonstrated by several mouse models. Targets of miR-125b include key proteins regulating apoptosis, innate immunity, inflammation and hematopoietic differentiation. Leukemia (2012) 26, 2011–2018; doi:10.1038/leu.2012.90 Keywords: microRNA; hematopoiesis; hematological malignancies; acute myeloid leukemia; acute lymphoblastic leukemia MicroRNAs (miRNAs) are 21–23-nucleotide non-coding RNAs that nucleotides with the seed region of miR-125b (ebv-miR-BART21-5p, have crucial roles in fundamental biological processes by ebv-miR-BART8 and rlcv-miR-rL1-25). In humans, as in most of the regulating the levels of multiple proteins. They are transcribed genomes, there are two paralogs (hsa-miR-125b-1 on chromosome as primary miRNAs and processed in the nucleus by the RNase III 11 and hsa-miR-125b-2 on chromosome 21), coding for the same endonuclease DROSHA to liberate 70-nucleotide stem loops, the mature sequence.
    [Show full text]
  • EIF2C2 Monoclonal Antibody (M01), Clone 2E12-1C9
    Produktinformation Diagnostik & molekulare Diagnostik Laborgeräte & Service Zellkultur & Verbrauchsmaterial Forschungsprodukte & Biochemikalien Weitere Information auf den folgenden Seiten! See the following pages for more information! Lieferung & Zahlungsart Lieferung: frei Haus Bestellung auf Rechnung SZABO-SCANDIC Lieferung: € 10,- HandelsgmbH & Co KG Erstbestellung Vorauskassa Quellenstraße 110, A-1100 Wien T. +43(0)1 489 3961-0 Zuschläge F. +43(0)1 489 3961-7 [email protected] • Mindermengenzuschlag www.szabo-scandic.com • Trockeneiszuschlag • Gefahrgutzuschlag linkedin.com/company/szaboscandic • Expressversand facebook.com/szaboscandic EIF2C2 monoclonal antibody (M01), clone 2E12-1C9 Catalog # : H00027161-M01 規格 : [ 100 ug ] List All Specification Application Image Product Mouse monoclonal antibody raised against a full length recombinant Western Blot (Cell lysate) Description: EIF2C2. Immunogen: EIF2C2 (AAH07633.1, 1 a.a. ~ 377 a.a) full-length recombinant protein with GST tag. MW of the GST tag alone is 26 KDa. Sequence: MPIQGQPCFCKYAQGADSVEPMFRHLKNTYAGLQLVVVILPGKTPVYAE VKRVGDTVLGMATQCVQMKNVQRTTPQTLSNLCLKINVKLGGVNNILLP enlarge QGRPPVFQQPVIFLGADVTHPPAGDGKKPSIAAVVGSMDAHPNRYCATV Western Blot (Transfected RVQQHRQEIIQDLAAMVRELLIQFYKSTRFKPTRIIFYRDGVSEGQFQQV lysate) LHHELLAIREACIKLEKDYQPGITFIVVQKRHHTRLFCTDKNERVGKSGNIP AGTTVDTKITHPTEFDFYLCSHAGIQGTSRPSHYHVLWDDNRFSSDELQI LTYQLCHTYVRCTRSVSIPAPAYYAHLVAFRARYHLVDKEHDSAEGSHTS GQSNGRDHQALAKAVQVHQDTLRTMYFA Host: Mouse enlarge Reactivity: Human Western Blot (Recombinant Isotype: IgG1 Kappa
    [Show full text]
  • Relative Contribution of Sequence and Structure Features to the Mrna Binding of Argonaute/ EIF2C–Mirna Complexes and the Degradation of Mirna Targets
    Downloaded from genome.cshlp.org on October 2, 2021 - Published by Cold Spring Harbor Laboratory Press Letter Relative contribution of sequence and structure features to the mRNA binding of Argonaute/ EIF2C–miRNA complexes and the degradation of miRNA targets Jean Hausser,1,3 Markus Landthaler,2,3,4 Lukasz Jaskiewicz,1 Dimos Gaidatzis,1,5 and Mihaela Zavolan1,6 1Biozentrum, University of Basel and Swiss Institute of Bioinformatics, CH-4056 Basel, Switzerland; 2Howard Hughes Medical Institute, Laboratory for RNA Biology, The Rockefeller University, New York, New York 10021, USA How miRNAs recognize their target sites is a puzzle that many experimental and computational studies aimed to solve. Several features, such as perfect pairing of the miRNA seed, additional pairing in the 39 region of the miRNA, relative position in the 39 UTR, and the A/Ucontent of the environment of the putative site, have been found to be relevant. Here we have used a large number of previously published data sets to assess the power that various sequence and structure features have in distinguishing between putative sites that do and those that do not appear to be functional. We found that although different data sets give widely different answers when it comes to ranking the relative importance of these features, the sites inferred from most transcriptomics experiments, as well as from comparative genomics, appear similar at this level. This suggests that miRNA target sites have been selected in evolution on their ability to trigger mRNA degradation. To understand at what step in the miRNA-induced response individual features play a role, we transfected human HEK293 cells with miRNAs and analyzed the association of Argonaute/EIF2C–miRNA complexes with target mRNAs and the degradation of these messages.
    [Show full text]
  • Dysregulated RNA-Induced Silencing Complex (RISC) Assembly Within CNS Corresponds with Abnormal Mirna Expression During Autoimmune Demyelination
    The Journal of Neuroscience, May 13, 2015 • 35(19):7521–7537 • 7521 Neurobiology of Disease Dysregulated RNA-Induced Silencing Complex (RISC) Assembly within CNS Corresponds with Abnormal miRNA Expression during Autoimmune Demyelination Przemysław Lewkowicz, Hanna Cwiklin´ska, Marcin P. Mycko, Maria Cichalewska, Małgorzata Domowicz, Natalia Lewkowicz, Anna Jurewicz, and Krzysztof W. Selmaj Department of Neurology, Laboratory of Neuroimmunology, Medical University of Lodz, 92-213 Lodz, Poland MicroRNAs (miRNAs) associate with Argonaute (Ago), GW182, and FXR1 proteins to form RNA-induced silencing complexes (RISCs). RISCs represent a critical checkpoint in the regulation and bioavailability of miRNAs. Recent studies have revealed dysregulation of miRNAs in multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE); however, the function of RISCs in EAE and MS is largely unknown. Here, we examined the expression of Ago, GW182, and FXR1 in CNS tissue, oligodendrocytes (OLs), brain-infiltrating T lymphocytes, and CD3 ϩsplenocytes (SCs) of EAE mic, and found that global RISC protein levels were signifi- cantly dysregulated. Specifically, Ago2 and FXR1 levels were decreased in OLs and brain-infiltrating T cells in EAE mice. Accordingly, assembly of Ago2/GW182/FXR1 complexes in EAE brain tissues was disrupted, as confirmed by immunoprecipitation experiments. In parallel with alterations in RISC complex content in OLs, we found downregulation of miRNAs essential for differentiation and survival of OLs and myelin synthesis. In brain-infiltrating T lymphocytes, aberrant RISC formation contributed to miRNA-dependent proinflam- matory helper T-cell polarization. In CD3 ϩ SCs, we found increased expression of both Ago2 and FXR1 in EAE compared with nonim- munized mice.
    [Show full text]
  • NIH Public Access Author Manuscript Epigenetics
    NIH Public Access Author Manuscript Epigenetics. Author manuscript; available in PMC 2009 June 23. NIH-PA Author ManuscriptPublished NIH-PA Author Manuscript in final edited NIH-PA Author Manuscript form as: Epigenetics. 2007 ; 2(2): 126±134. Epigenetics and Neural Developmental Disorders: Washington DC, September 18 and 19, 2006 Xinyu Zhao1,*, ChangHui Pak2, Richard D. Smrt1, and Peng Jin2,* 1 Department of Neuroscience; University of New Mexico School of Medicine; Albuquerque, New Mexico USA 2 Department of Human Genetics; Emory University School of Medicine; Atlanta, Georgia USA Abstract Neural developmental disorders, such as autism, Rett Syndrome, Fragile X syndrome, and Angelman syndrome manifest during early postnatal neural development. Although the genes responsible for some of these disorders have been identified, how the mutations of these genes affect neural development is currently unclear. Emerging evidence suggest that these disorders share common underlying defects in neuronal morphology, synaptic connectivity and brain plasticity. In particular, alterations in dendritic branching and spine morphology play a central role in the pathophysiology of most mental retardation disorders, suggesting that common pathways regulating neuronal function may be affected. Epigenetic modulations, mediated by DNA methylation, RNA-associated silencing, and histone modification, can serve as an intermediate process that imprints dynamic environmental experiences on the “fixed” genome, resulting in stable alterations in phenotypes. Disturbance in epigenetic regulations can lead to inappropriate expression or silencing of genes, causing an array of multi-system disorders and neoplasias. Rett syndrome, the most common form of mental retardation in young girls, is due to l mutation of MECP2, encoding a methylated DNA binding protein that translates DNA methylation into gene repression.
    [Show full text]
  • T Lymphocytes Mrna, and Protein Expression in Activated Networks
    MicroRNA Regulation of Molecular Networks Mapped by Global MicroRNA, mRNA, and Protein Expression in Activated T Lymphocytes This information is current as of September 23, 2021. Yevgeniy A. Grigoryev, Sunil M. Kurian, Traver Hart, Aleksey A. Nakorchevsky, Caifu Chen, Daniel Campbell, Steven R. Head, John R. Yates III and Daniel R. Salomon J Immunol 2011; 187:2233-2243; Prepublished online 25 July 2011; Downloaded from doi: 10.4049/jimmunol.1101233 http://www.jimmunol.org/content/187/5/2233 http://www.jimmunol.org/ Supplementary http://www.jimmunol.org/content/suppl/2011/07/25/jimmunol.110123 Material 3.DC1 References This article cites 83 articles, 31 of which you can access for free at: http://www.jimmunol.org/content/187/5/2233.full#ref-list-1 Why The JI? Submit online. by guest on September 23, 2021 • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2011 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology MicroRNA Regulation of Molecular Networks Mapped by Global MicroRNA, mRNA, and Protein Expression in Activated T Lymphocytes Yevgeniy A.
    [Show full text]
  • A Drosophila Fragile X Protein Interacts with Components of Rnai and Ribosomal Proteins
    Downloaded from genesdev.cshlp.org on September 25, 2021 - Published by Cold Spring Harbor Laboratory Press A Drosophila fragile X protein interacts with components of RNAi and ribosomal proteins Akira Ishizuka,1,2,3 Mikiko C. Siomi,1,3 and Haruhiko Siomi1,4 1Institute for Genome Research, 2Graduate School of Nutrition University of Tokushima, Tokushima 770-8503, Japan Fragile X syndrome is a common form of inherited mental retardation caused by the loss of FMR1 expression. The FMR1 gene encodes an RNA-binding protein that associates with translating ribosomes and acts as a negative translational regulator. In Drosophila, the fly homolog of the FMR1 protein (dFMR1) binds to and represses the translation of an mRNA encoding of the microtuble-associated protein Futsch. We have isolated a dFMR1-associated complex that includes two ribosomal proteins, L5 and L11, along with 5S RNA. The dFMR1 complex also contains Argonaute2 (AGO2) and a Drosophila homolog of p68 RNA helicase (Dmp68). AGO2 is an essential component for the RNA-induced silencing complex (RISC), a sequence-specific nuclease complex that mediates RNA interference (RNAi) in Drosophila. We show that Dmp68 is also required for efficient RNAi. We further show that dFMR1 is associated with Dicer, another essential component of the RNAi pathway, and microRNAs (miRNAs) in vivo, suggesting that dFMR1 is part of the RNAi-related apparatus. Our findings suggest a model in which the RNAi and dFMR1-mediated translational control pathways intersect in Drosophila. Our findings also raise the possibility that defects in an RNAi-related machinery may cause human disease. [Keywords: fragile X syndrome; FMR1; RNAi; RNA helicase; miRNA] Received July 9, 2002; revised version accepted August 22, 2002.
    [Show full text]
  • High-Risk Myeloma Is Associated with Global Elevation of Mirnas and Overexpression of EIF2C2/AGO2
    High-risk myeloma is associated with global elevation of miRNAs and overexpression of EIF2C2/AGO2 Yiming Zhoua,1, Lijuan Chena,b,1, Bart Barlogiea, Owen Stephensa, Xiaosong Wua, David R. Williamsa, Marie-Astrid Cartrona, Frits van Rheea, Bijay Naira, Sarah Waheeda, Mauricio Pineda-Romana, Yazan Alsayeda, Elias Anaissiea, and John D. Shaughnessy, Jr.a,2 aDonna D. and Donald M. Lambert Laboratory for Myeloma Genetics, Myeloma Institute for Research and Therapy, University of Arkansas for Medical Sciences, Little Rock, AR 72205; and bDepartment of Hematology, First Affiliated Hospital of Nanjing Medical University, Jiangsu Province Hospital, Nanjing, 210029,China Edited* by Carlo M. Croce, Ohio State University, Columbus, OH, and approved March 17, 2010 (received for review July 30, 2009) MicroRNAs (miRNAs) are noncoding RNAs that regulate global gene liferation in MM cell lines, and Lionetti et al. (14) identified 16 expression. miRNAs often act synergistically to repress target genes, miRNAs sensitive to DNA copy number. and their dysregulation can contribute to the initiation and pro- To further investigate the potential involvement of miRNA gression of a variety of cancers. The clinical relationship between in MM, we performed integrative analyses of both miRNA global expression of miRNA and mRNA in cancer has not been expression profiles and protein-coding gene expression profiles studied in detail. We used whole-genome microarray analyses of (GEPs) of myeloma cells from newly diagnosed patients. Global CD138-enriched plasma cells from 52 newly diagnosed cases of increases in miRNA expression were seen in cases with high-risk multiple myeloma to correlate miRNA expression profiles with a MM, which sharply contrasts with discriminate expression validated mRNA-based risk stratification score, proliferation index, changes of only selected miRNAs that are seen in other cancers and predefined gene sets.
    [Show full text]
  • Integrative Modeling and Visualization of Exosomes 7
    Integrative Modeling and Visualization of Exosomes 7 Integrative Modeling and Visualization of Exosomes Julia Jiménez, Ludovic Autin, Inmaculada Ibáñez de Cáceres, David S. Goodsell As part of this work, we have used small, well-defined systems to Information from proteomics, microscopy, and structural test and apply new methods. Because the work is highly biology are integrated to create structural models of exosomes, small vesicles released from cells. Three visualization methods integrative, a wide variety of data must be gathered (or are employed and compared: 2D painting of a cross section estimated) in order to generate a convincing model. Early using traditional media, manual creation of a cross section illustrative work focused on well-studied systems such as red using the mesoscale 2.5D digital painting software cellPAINT, blood cells and Escherichia coli (Goodsell 1992). For our 3D and generation of a 3D atomic model using the mesoscale modeling work, we have used simpler systems, including HIV modeling program cellPACK. (Johnson et al. 2014) and mycoplasma bacteria (Goodsell, Autin, et al. 2018). Exosomes provide an excellent test case for current mesoscale modeling methods. Sufficient proteomic, structural, and ultrastructural data are available to define a convincing model, Keywords however, there are still grey areas in this knowledge that may Exosome, integrative structural biology, molecular benefit from mesoscale modeling. Given their growing role in visualization, non-photorealistic rendering, protein structure many disease states, many illustrations of exosomes have been created, ranging from simple schematics to highly rendered editorial and advertising imagery (an internet search of Introduction “exosome” will yield hundreds of examples). However, to our Modeling of the molecular structure of cells is rapidly moving knowledge, no detailed integrative models of exosomes have from an art into a science.
    [Show full text]
  • Recombinant AGO2 Protein Catalog No: 31486, 31886 Quantity: 20, 1000 Μg Lot No: 07315001 Concentration: 0.4 Expressed In: Baculovirus Μg/Μl Source: Human
    Recombinant AGO2 protein Catalog No: 31486, 31886 Quantity: 20, 1000 µg Lot No: 07315001 Concentration: 0.4 Expressed In: Baculovirus µg/µl Source: Human Buffer Contents: Full length recombinant AGO2 protein expressed in Sf9 cells and is provided in 25 mM HEPES, pH 7.5, 500 mM NaCl, 5% glycerol, 0.04% Triton X-100, 0.2 mM TCEP and 0.2 mg/ml 3X FLAG peptide. Background: AGO2 (Argonaute RISC Catalytic Component 2, also known as Q10; EIF2C2) is a member of the Argonaute family of proteins which play a role in RNA interference. This process is important both in gene regulation by microRNAs and in defense against viral infections. The RNA-induced silencing complex, or RISC, is a multiprotein complex that includes argonaute proteins and one strand of a small interfering RNA (siRNA) or microRNA (miRNA). RISC uses the siRNA or miRNA as a template for recognizing complementary mRNA. When it recognizes a complementary strand, it activates AGO2 to cleave the mRNA. The precise mechanism of gene silencing depends on the degree of complementarity between the miRNA or siRNA and its target. Binding of RISC to a perfectly complementary mRNA generally results in silencing due to endonucleolytic cleavage of the mRNA specifically by AGO2. Binding of RISC to a partially complementary mRNA results in silencing through inhibition of translation, and this is independent of endonuclease activity. In some cases, RISC-mediated translational repression is also observed for miRNAs that perfectly match the 3’ untranslated region (3’UTR). In addition, AGO2 can also up-regulate the posttranscriptional upregulation of specific mRNAs under certain growth conditions.
    [Show full text]